Method for improving purity of polypeptide in soybean enzymolysis product
Ultrasonic-assisted magnesium sulfate precipitation of soy protein gels has been solved, and the problem of low purity of peptides in enzymatic lysis is improved, and the purity of peptides in soy enzyme solution is achieved, thereby achieving higher mass concentration.
Patent Information
- Application Number
- CN202411689328.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-25
- Publication Date
- 2025-01-10
AI Technical Summary
When soy protein is prepared by enzymatic method, high temperature enzyme depletion causes the viscosity of the soy protein solution that has not been enzymatically dissolved increases, making it difficult to separate, resulting in a low purity of the peptide.
Ultrasonic-assisted magnesium sulfate precipitated soy protein gel is used to improve the purity of the peptide in the soy enzyme solution product. Specific steps include enzymatic decomposition of soy protein and ultrasonic assisted magnesium sulfate treatment, ultrasonic power is 50-500W, time is 10-90 minutes, followed by centrifugation and lyophilization to obtain a high-purity polypeptide.
The mass concentration of the polypeptide in the soybean enzymatic product was improved, and the enzymatic analysis of the polypeptide was increased by 3.03-7.74% compared with the use of alkaline protease, and 1.22-5.01% compared with the use of magnesium sulfate precipitation, which significantly improved the purity of the polypeptide.
Abstract
Description
Technical Field
[0001] The invention belongs to the field of polypeptide purification, and particularly relates to a method for improving the purity of polypeptides in soybean enzymatic hydrolysis products. Background Art
[0002] As a common source of high-quality plant protein, soy protein contains a variety of essential amino acids for the human body and has high nutritional value. Studies have shown that soy peptides obtained after hydrolysis of soy protein have better physical and chemical properties such as solubility, stability, and absorbability, as well as biological activities such as hypoallergenicity, antioxidant, and cholesterol-lowering. Therefore, the hydrolysis of soy protein to prepare peptides is conducive to expanding its application range and improving its high-value utilization, and promoting the development and utilization of peptide products.
[0003] At present, the hydrolysis methods of soybean protein mainly include acid method, alkali method and enzymatic method. Enzymatic method is the most commonly used method for hydrolysis of soybean protein due to its mild reaction and green environmental protection. However, when using enzymatic method to prepare soybean peptides, high temperature will induce the viscosity of the unhydrolyzed soybean protein solution to increase while killing the enzyme, which is not easy to separate, resulting in low purity of peptides in the obtained soybean enzymatic product (Yu Bo. Research on the production of soybean active peptides by fermenting soybean meal with Bacillus subtilis [D]. Nanjing, Jiangsu: Nanjing Agricultural University, 2006.). Therefore, physical and chemical methods can be used to change this phenomenon, so that more soybean protein is precipitated and the purity of peptides in soybean enzymatic products is improved.
[0004] Existing studies have shown that heat-denatured soy protein will produce salting-out after adding salt, which promotes protein aggregation. Ultrasound, as a green and efficient protein modification technology, can be used to try to use ultrasound-assisted magnesium sulfate precipitation of soy protein in the enzymatic hydrolysis product to improve the purity of polypeptides in the soy enzymatic hydrolysis product. Summary of the invention
[0005] The purpose of the present invention is to provide a method for improving the purity of polypeptides in soybean enzymatic hydrolysates, specifically, on the basis of enzymatic hydrolysis of soybean protein with alkaline protease, ultrasonic-assisted magnesium sulfate precipitation of soybean protein gel is used to improve the purity of polypeptides in soybean enzymatic hydrolysates.
[0006] In order to achieve the above-mentioned purpose of the invention, the specific technical scheme is as follows: A method for improving the purity of polypeptides in soybean enzymatic hydrolysates is carried out according to the following steps:
[0007] (1) Enzymatic hydrolysis of soybean protein: a certain amount of soybean protein was dissolved in pure water to prepare a soybean protein suspension with a mass concentration of 50 g / L. After adjusting the pH to 8.5 with a 4 mol / L sodium hydroxide solution, 5% (based on the mass of soybean protein) alkaline protease was added. The enzymatic hydrolysis was carried out at a hydrolysis temperature of 50° C. and a hydrolysis pH of 8.5 for 10-360 min. After the enzymatic hydrolysis was completed, the enzymatic hydrolyzate was collected and the enzyme was inactivated in a 95° C. water bath for 15 min to obtain a soybean protein hydrolyzate product.
[0008] (2) Ultrasonic-assisted magnesium sulfate to improve the purity of polypeptides in soybean enzymatic hydrolysate: Immediately collect the soybean protein hydrolysate after the enzyme inactivation in step (1), add magnesium sulfate to make the final concentration 3g / L, cool to room temperature and then perform ultrasonic treatment. The ultrasonic treatment parameters are as follows: ultrasonic power is 50-500W, and ultrasonic time is 10-90min. After the ultrasonic treatment, collect the soybean protein hydrolysate, centrifuge at 12000r / min for 10min, collect the supernatant and freeze-dry to obtain a soybean protein hydrolysate containing high-purity polypeptides.
[0009] The invention has the advantages that: the invention uses ultrasound-assisted magnesium sulfate to improve the purity of polypeptides in soybean enzymatic hydrolysis products, and the mass concentration of polypeptides in the obtained soybean enzymatic hydrolysis products is 2.51-2.57 g / L, which is 3.03-7.74% higher than that of alkaline protease hydrolysis alone, and is 1.22-5.01% higher than that of soybean protein precipitated by magnesium sulfate. DETAILED DESCRIPTION
[0010] The technical solution of the present invention is further described in detail below through embodiments.
[0011] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific embodiments and reference data. It should be understood that these embodiments are only further explanations of the present invention and cannot be construed as limiting the scope of protection of the present invention. Technical engineers in this field can make further improvements and adjustments to the present invention based on the above invention content.
[0012] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The sources, trade names and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified.
[0013] The alkaline protease (enzyme activity 54680 U / g) in this example and the control example was purchased from Beijing Gaoruisen Technology Co., Ltd.
[0014] The ultrasonic equipment used in this embodiment and the comparative example is a KS-500DV liquid crystal ultrasonic cleaner purchased from Kunshan Jielimei Ultrasonic Instrument Co., Ltd.
[0015] The method for determining the polypeptide concentration in this example and the control example is as follows: 0.1 g of sample is weighed and dissolved in 4 mL of pure water, and the polypeptide mass concentration in the supernatant is determined by the Folin phenol method after protein precipitation with 10% trichloroacetic acid.
[0016] The above soybean protein hydrolysate is an enzymatic hydrolysate with a higher degree of hydrolysis, and can also be simply replaced by an enzymatic hydrolysate with a higher degree of hydrolysis prepared from other protein raw materials.
[0017] Comparative Example 1
[0018] Weigh 50g of soy protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 10min at a hydrolysis temperature of 50°C and a hydrolysis pH of 8.5. After the hydrolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min, then centrifuge at 12000r / min for 10min, collect the supernatant and freeze-dry it, and the polypeptide mass concentration is determined to be 2.37g / L.
[0019] Comparative Example 2
[0020] Weigh 50g of soy protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 360min at a hydrolysis temperature of 50°C and a hydrolysis pH of 8.5. After the hydrolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min, then centrifuge at 12000r / min for 10min, collect the supernatant and freeze-dry it, and the polypeptide mass concentration is determined to be 2.47g / L.
[0021] Comparative Example 3
[0022] Weigh 50g of soy protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 10min at a hydrolysis temperature of 50°C and a hydrolysis pH of 8.5. After the hydrolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min. Immediately after the completion, add magnesium sulfate to make a final concentration of 3g / L. After cooling to room temperature, centrifuge at 12000r / min for 10min. After collecting the supernatant and lyophilizing it, the polypeptide mass concentration is determined to be 2.43g / L, which is 2.60% higher than that of enzymolysis with alkaline protease alone.
[0023] Comparative Example 4
[0024] Weigh 50g of soy protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 360min at a hydrolysis temperature of 50°C and a hydrolysis pH of 8.5. After the hydrolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min. Immediately after the hydrolysis, add magnesium sulfate to make a final concentration of 3g / L. After cooling to room temperature, centrifuge at 12000r / min for 10min. After collecting the supernatant and lyophilizing it, the polypeptide mass concentration is determined to be 2.51g / L, which is 1.79% higher than that of enzymolysis with alkaline protease alone.
[0025] Example 1
[0026] Weigh 50g of soybean protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 10min under the conditions of enzymolysis temperature of 50°C and enzymolysis pH of 8.5. After the enzymolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min. After the enzymolysis is completed, add magnesium sulfate immediately to make the final concentration of 3g / L. After cooling to room temperature, ultrasonic treatment is performed on the soybean polypeptide solution, and the ultrasonic power is set to 50W and the ultrasonic time is 10min. After the ultrasonic treatment, centrifuge at 12000r / min for 10min. After collecting the supernatant and lyophilizing it, the polypeptide mass concentration is determined to be 2.53g / L, which is 6.57% higher than that of enzymolysis with alkaline protease alone and 3.87% higher than that of soybean protein precipitated with magnesium sulfate.
[0027] Example 2
[0028] Weigh 50g of soybean protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 10min under the conditions of enzymolysis temperature of 50°C and enzymolysis pH of 8.5. After the enzymolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min. After the enzymolysis is completed, add magnesium sulfate immediately to make the final concentration of 3g / L. After cooling to room temperature, ultrasonic treatment is performed on the soybean polypeptide solution, and the ultrasonic power is set to 50W and the ultrasonic time is set to 90min. After the ultrasonic treatment, centrifuge at 12000r / min for 10min. After collecting the supernatant and lyophilizing it, the polypeptide mass concentration is determined to be 2.52g / L, which is 6.44% higher than that of enzymolysis with alkaline protease alone and 3.75% higher than that of soybean protein precipitated with magnesium sulfate.
[0029] Example 3
[0030] Weigh 50g of soybean protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 10min under the conditions of enzymolysis temperature of 50°C and enzymolysis pH of 8.5. After the enzymolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min. After the end, add magnesium sulfate immediately to make the final concentration of 3g / L. After cooling to room temperature, ultrasonic treatment is performed on the soybean polypeptide solution, and the ultrasonic power is set to 500W and the ultrasonic time is 10min. After the end of the ultrasonic treatment, centrifuge at 12000r / min for 10min. After collecting the supernatant and lyophilizing it, the polypeptide mass concentration is determined to be 2.55g / L, which is 7.74% higher than that of enzymolysis with alkaline protease alone and 5.01% higher than that of soybean protein precipitated with magnesium sulfate.
[0031] Example 4
[0032] Weigh 50g of soybean protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 10min under the conditions of enzymolysis temperature of 50°C and enzymolysis pH of 8.5. After the enzymolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min. After the end, add magnesium sulfate immediately to make the final concentration of 3g / L. After cooling to room temperature, ultrasonic treatment is performed on the soybean polypeptide solution, and the ultrasonic power is set to 500W and the ultrasonic time is set to 90min. After the end of the ultrasonic treatment, centrifuge at 12000r / min for 10min. After collecting the supernatant and lyophilizing it, the polypeptide mass concentration is determined to be 2.51g / L, which is 5.93% higher than that of enzymolysis with alkaline protease alone and 3.25% higher than that of soybean protein precipitated with magnesium sulfate.
[0033] Example 5
[0034] Weigh 50g of soybean protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 360min under the conditions of enzymolysis temperature of 50°C and enzymolysis pH of 8.5. After the enzymolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min. After the completion, add magnesium sulfate immediately to make the final concentration of 3g / L. After cooling to room temperature, ultrasonic treatment is performed on the soybean polypeptide solution, and the ultrasonic power is set to 50W and the ultrasonic time is 10min. After the completion of the ultrasonic treatment, centrifuge at 12000r / min for 10min. After the supernatant is collected and freeze-dried, the polypeptide mass concentration is determined to be 2.55g / L, which is 3.03% higher than that of enzymolysis with alkaline protease alone and 1.22% higher than that of soybean protein precipitated with magnesium sulfate.
[0035] Example 6
[0036] Weigh 50g of soybean protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 360min under the conditions of enzymolysis temperature of 50°C and enzymolysis pH of 8.5. After the enzymolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min. After the completion, add magnesium sulfate immediately to make the final concentration of 3g / L. After cooling to room temperature, ultrasonic treatment is performed on the soybean polypeptide solution, and the ultrasonic power is set to 50W and the ultrasonic time is set to 90min. After the completion of the ultrasonic treatment, centrifuge at 12000r / min for 10min. After the supernatant is collected and freeze-dried, the polypeptide mass concentration is determined to be 2.57g / L, which is 4.10% higher than that of enzymolysis with alkaline protease alone and 2.27% higher than that of soybean protein precipitated with magnesium sulfate.
[0037] Example 7
[0038] Weigh 50g of soybean protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 360min under the conditions of enzymolysis temperature of 50°C and enzymolysis pH of 8.5. After the enzymolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min. After the end, add magnesium sulfate immediately to make the final concentration of 3g / L. After cooling to room temperature, ultrasonic treatment is performed on the soybean polypeptide solution, and the ultrasonic power is set to 500W and the ultrasonic time is 10min. After the end of the ultrasound, centrifuge at 12000r / min for 10min. After collecting the supernatant and lyophilizing it, the polypeptide mass concentration is determined to be 2.57g / L, which is 4.15% higher than that of enzymolysis with alkaline protease alone and 2.32% higher than that of soybean protein precipitated with magnesium sulfate.
[0039] Example 8
[0040] Weigh 50g of soybean protein, add 1000mL of distilled water, adjust the pH to 8.5 with 4mol / L sodium hydroxide solution, add 5% (E / S) alkaline protease, and perform enzymolysis for 360min under the conditions of enzymolysis temperature of 50°C and enzymolysis pH of 8.5. After the enzymolysis is completed, collect the enzymolysis solution and inactivate the enzyme in a 95°C water bath for 15min. After the completion, add magnesium sulfate immediately to make the final concentration of 3g / L. After cooling to room temperature, ultrasonic treatment is performed on the soybean polypeptide solution, and the ultrasonic power is set to 500W and the ultrasonic time is set to 90min. After the completion of the ultrasonic treatment, centrifuge at 12000r / min for 10min. After the supernatant is collected and freeze-dried, the polypeptide mass concentration is determined to be 2.56g / L, which is 3.52% higher than that of enzymolysis with alkaline protease alone and 1.70% higher than that of soybean protein precipitated with magnesium sulfate.
Claims
1. A method for improving the purity of polypeptides in soybean enzymatic hydrolysates, characterized in that Follow the steps below: (1) Enzymatic hydrolysis of soybean protein: a certain amount of soybean protein was dissolved in pure water to prepare a soybean protein suspension with a mass concentration of 50 g / L, the pH was adjusted to 8.5 with a 4 mol / L sodium hydroxide solution, 5% (based on the protein mass of soybean protein) alkaline protease was added, and the enzymatic hydrolysis was carried out at a hydrolysis temperature of 50° C. and a hydrolysis pH of 8.5 for 10-360 min. After the enzymatic hydrolysis was completed, the enzymatic hydrolyzate was collected and the enzyme was inactivated in a 95° C. water bath for 15 min to obtain a soybean protein hydrolyzate product; (2) Ultrasonic-assisted magnesium sulfate to improve the purity of polypeptides in soybean enzymatic hydrolysate: Immediately collect the soybean protein hydrolysate after the enzyme inactivation in step (1), add magnesium sulfate to make the final concentration of 3 g / L, cool to room temperature and then perform ultrasonic treatment. After the ultrasonic treatment, collect the soybean protein hydrolysate, centrifuge at 12000 r / min for 10 min, collect the supernatant and freeze-dry to obtain a soybean protein hydrolysate containing high-purity polypeptides.
2. A method for improving the purity of polypeptides in soybean enzymatic hydrolysates according to claim 1, characterized in that The ultrasonic treatment parameters in step (2) are as follows: the ultrasonic power is 50-500W.
3. A method for improving the purity of polypeptides in soybean enzymatic hydrolysates according to claim 1, characterized in that The ultrasonic treatment parameters in step (2) are as follows: the ultrasonic time is 10-90 min.